UNIVERSITA’ DEGLI STUDI DI ROMA
“TOR VERGATA”
DOTTORATO DI RICERCA
“BIOLOGIA CELLULARE E MOLECOLARE”
2007 - XIX ciclo
Identification of novel and direct target genes of p73
ABSTRACT
The p53 paralogues p73, p63 and their respective truncated isoforms have been shown to be critical regulators of developmental and differentiation processes. Indeed, both p73 and p63 deficient mice exhibit severe developmental defects. Here, we show that the S100A2 gene, whose transcript and protein are induced during keratinocyte differentiation of HaCaT cells, is a direct transcriptional target of p73β and ΔNp63α and is required for proper keratinocyte differentiation. Transactivation assays reveal that p73β and ΔNp63α exert opposite transcriptional effects on the S100A2 gene. While ΔNp63α is found in vivo onto S100A2 regulatory regions predominantly in proliferating cells, p73β is recruited in differentiating cells. Silencing of p73 impairs the induction of S100A2 during the differentiation of HaCaT cells. Moreover, silencing of p73 or S100A2 impairs the proper expression of keratinocyte differentiation markers. Of note, p53 family members do not trigger S100A2 gene expression in response to apoptotic doses of cisplatin and doxorubicin. The p53 family is also known to be involved in the transcriptional control of growth arrest and apoptosis. Despite the recent identification of specific p73-target genes by genome-wide expression profile techniques, p73-mediated apoptosis occurs mostly through the activation of a set of genes that were originally found to be activated by p53. This suggests that promoter selectivity by both p53 and p73 might be the result of biochemical events such as post-translational modifications and specific protein-protein interactions.
The transcriptional coactivator Yes-associated protein (YAP) has been demonstrated to interact with and to enhance p73-dependent apoptosis in response to DNA damage. Here we show the existence of specific target genes whose transcriptional activation during the apoptotic response requires both p73 and YAP. In particular p73 and YAP are concomitantly recruited onto the regulatory regions of the promyelocytic leukemia gene (PML); an essential event for PML induction after cisplatin treatment. Moreover, sequestring YAP into the cytoplasm by a constitutively active mutant of AKT leads to a reduction of p300 recruitment onto the PML regulatory regions which correlates with a reduction in histone acetylation and a
To my family
CONTENTS
INTRODUCTION 3
p53 family 4
p63 and p73 play important roles in development and differentiation 8
p63 and p73 expression in normal human tissues 10
Transcriptional and apoptotic activity of p63 and p73 11
Regulation of p73 protein stability and transcriptional activity 13
Post-translational modifications during activation 16
Alteration of p73 expression in human cancer 18
Alteration of p63 expression in human cancer 20
Transcriptional regulation of the main promoter of p73 20
Mutual regulation between p73 and p53 22
An autoregulatory feedback loop exists among p53, TAp73 and ΔNp63 24 p73 and chemosensitivity 24
YAP 26
S100 proteins 30
Intracellular roles of S100 proteins 32
Inhibition of protein phosphorylation 32
Regulation of enzyme activity 33
Regulation of cell growth and differentiation 34
S100 proteins and the cytoskeleton 35
Extracellular roles of S100 proteins 35
S100 proteins and cancer 35
S100 proteins in the epidermis 36
PML 38
Role of PML in multiple apoptotic pathways 43
PML and p53 44
PML and p73 45
Cell culture 52
Immunoprecipitation and Western Blot Analysis 52
Indirect immunofluorescence 53
Plasmids 53
Transfections and luciferase assays 54
RNA extraction and reverse transcriptase reaction 54
Real-Time RT-PCR 55
Formaldehyde cross-linking and chromatin immunoprecipitation 55
Electrophoretic mobility shift assay 56
siRNA studies 57
Cell cycle analysis 57
RESULTS 58
S100A2 gene is a direct transcriptional target of p53 homologues during keratinocyte differentiation. 59
S100A2 expression is induced during keratinocyte differentiation. 59
S100A2 is a direct transcriptional target of p73β and ΔNp63α. 59
In vivo recruitment of p73β and ΔNp63α onto the regulatory regions of S100A2 gene. 62
Silencing of p73 expression impairs S100A2 upregulation during keratinocyte differentiation. 64
Silencing of S100A2 impairs keratinocyte differentiation. 67
S100A2 gene is not induced in response to DNA damage. 67
The protein complex p73/YAP is a transcriptional regulator of PML. 73 Search for genes modulated by the protein complex p73/YAP. 73
p73 and YAP are required for PML induction after treatment with cisplatin. 73
PML is induced by p73. 78
PML is a direct transcriptional target of p73 and YAP. 81
YAP and PML physically interact in vivo. 81
YAP degradation occurs through the ubiquitin-proteasome pathway and is negatively regulated by PML. 84
DISCUSSION 88
p53 family
p53 was first observed to co-immunoprecipitate with the large and small T antigens in Simian virus 40-transformed cells (Kress et al., 1979; Lane and Crawford, 1979; Linzer and Levine, 1979). Since that time, p53 has evolved from a potential oncogene to the principal tumour suppressor in mammals: its inactivation is a precondition to most human cancer. Further confirmation of the role of p53 in tumour suppression has come from animal models that show increased tumorigenesis in p53-null mice (Donehower et al., 1992). p53 has been recognized as a guardian against cellular stressors, particularly those that inflict DNA damage. It is a transcription factor which exerts its protective effects by inducing cell-cycle arrest to allow repair processes or, falling that, by promoting cellular senescence or apoptosis (Levine, 1997).
Although p53 was long considered to be unique, two novel family members were identified and termed p73 and p63 (Kaghad et al., 1997; Yang et al., 1998). Both genes give rise to proteins that have both entirely novel functions and p53-related functions. The gene structure of p53, p63 and p73 is highly conserved from mollusk to human. The three most conserved domains in all three genes are the N-terminal transactivation domain, the central DNA binding domain and the C-terminal oligomerization domain. Both p63 and p73 share >60% amino acid identity with the DNA binding region of p53 (and even higher identity among themselves), including conservation of all DNA contact and structural residues that are hotspots for p53 mutations in human tumours. In addition, p73 shows 38% identity with the p53 tetramerization domain and 29% identity with the p53 transactivation domain. In vertebrates, the p73 and p63 genes are ancestral to p53 and possibly evolved from a common p63/p73 archetype (Kaghad et al., 1997; Yang et al., 1998).
In addition to their similar protein structure, the three members are prone to alternative splicing and their transcription is driven by alternative promoters, giving rise to a complex expression of proteins. So far, three p53 proteins were identified: the full length, the Δ40p53 (also known as ΔNp53 or p47) lacks the first 40 amino acids resulting from either an alternative splicing of the intron 2 or an alternative initiation of translation and p53i9 which results from an alternative splicing of intron 9 and lack the last 60 amino acids. Δ40p53 partially possesses the transactivation domain and it is therefore able to transactivate p53 target genes but mainly acts as a dominant negative inhibitor of the full length protein (Courtois et al., 2002; Ghosh et
binding ability (Flaman et al., 1996). However, recent investigations using the RACE technique showed that p53 gene structure is similar to its counterpart members (Bourdon et al., 2005). p53 also possesses a second promoter located within intron 4 from which the mRNA transcript gives rise to a protein lacking the first 133 amino acids and to two C-terminal splice variant proteins lacking the tetramerization domain. The first promoter generates six proteins including the full length, two proteins lacking the tetramerization domain (p53β and γ) and three Δ40p53 proteins (Δ40p53, Δ40p53β and Δ40p53γ).
p63 and p73 have two promoters: P1 in the 5' untranslated region upstream of the noncoding exon 1 and P2 within the 23kb spanning intron 3. P1 and P2 promoters produce two diametricallyopposing classes of proteins: those containing the TA (TAp63and TAp73) and those lacking it (ΔNp63 and ΔNp73). ΔNp63 and ΔNp73occur in human and mouse. In addition, alternative exon splicing of the P1 transcripts of p63 and p73 give rise to other isoformslacking the transactivation domain (e.g., ΔN'p73, Ex2Delp73, and Ex2/3Delp73; Kaghad et al., 1997; Stiewe et al., 2002; Fillippovich et
al., 2001; Ishimoto et al., 2002). Of importance, the ΔNp73and ΔN'p73 transcripts encode the same protein due to the useof a second translational start site because of an upstreampremature stop in ΔN'p73 (Ishimoto et al., 2002). TA proteins mimic p53 function in cell culture including transactivating many p53 target genesand inducing apoptosis, whereas (the collectively called) ΔTA proteins act as dominant-negative inhibitors of themselves and of other family members in vivo in the mouse and in transfectedhuman cells (Yang et al., 1998; Yang et al., 2000; Pozniak et al., 2000). Strikingly, the p63 locus is containedwithin a frequently amplified region in squamous cell carcinoma (Hibi et al., 2000), and squamous epithelium of the skin and squamous carcinoma produce high levels of ΔNp6 (also called p68AIS). Furthermore, ΔNp73 is the predominant p73 product in the developing mouse nervous system and is required to counteract the proapoptoticaction of p53 (Yang et al., 2000; Pozniak et al., 2000).
Additional complexity is generated at the COOH terminus: p73 and p63 undergo multiple COOH-terminal splicings of exons 10to 14, skipping one or several exons. Thus far, nine transcripts were found for p73:
(Stiewe et al., 2002; Ishimoto et al., 2002). In some COOH-terminal isoforms, exon splicing also leads to unique sequences due to frameshifts. For p63,three isotypes (α, β and γ) are made. Splicing of different "tails" further modulates the p53-like function of TA proteins,although they do not appear to vary much in their role in tumorigenesis.Structurally, the γ forms of p73 and p63 most closely resemble p53 itself, harboring just a small COOH-terminal extension beyond the last 30-amino acid stretch of p53. Surprisingly, whereasTAp63γ (also called p51A) is as powerful as p53 in transactivation and apoptosis assays (Yang et al., 1998), TAp73γ is rather weak. The αforms ofp73 and p63 contain an additional highly conserved
sterileαmotif (SAM). SAMs are protein-protein interaction modules found in a wide variety of proteins implicated in development. Inaddition, the p73 SAM domain can bind to anionic and zwitterioniclipid membranes (Barrera
et al., 2003). The crystal and solution structures ofp73 SAM agree with each other and feature a five-helix bundlefold that is characteristic of all SAM domain structures (Chi et al., 1999; Wang et al., 2001). Other SAM-containing proteins are the ETS transcriptionfactor TEL that plays a role in leukemia, the polycomb group of homeotic transcription factors, and the ephrin receptors.Despite predictions of homo- and hetero-oligomerization of SAM-containing proteins, p73 SAM appears monomeric by experimental analysis,casting doubt whether this domain mediates interaction of p73with heterologous proteins (Wang et al., 2001). There are also functional differencesbetween TAp73α and TAp63α. Whereas TAp73α is comparable with p53in potency in transactivation and apoptosis assays, TAp63α (also called p51B) is very weak (Yang et al., 1998). One reason for this difference could be that p63α isoforms contain a 27-kDa COOH-terminal regionthat drastically reduces its transcriptional activity (Serber et al., 2002). This domain is necessary and sufficient for transcriptionalinhibition and acts by binding to a region in the NH2-terminalTA of p63, which is homologous to the MDM2 binding site in p53. Of note, this transactivation inhibitory domain is biologicallyimportant, because patients with deletions in this p63 domainhave phenotypes very similar to patients with mutations in theDBD (Serber et al., 2002).
Fig.1. Structure of the human p53, p63, and p73 genes.
The p53 family includes the three genes p53, p63 and p73. They have a modular structure consisting of the transactivation domain (TA), the DNA binding domain (DBD) and the oligomerization domain. All genes are expressed as two major types: full-length proteins containing the TA domain and ΔN proteins missing the TA domain. In addition, extensive COOH-terminal splicing further modulate the p53-like functions of the TA proteins.
p63 and p73 play important roles in development and differentiation Both genes play important and, despite their structural similarity, surprisingly unique roles in mouse and human development. This is powerfully revealed by the striking developmental phenotypesof p63- and p73-deficient mice (Yang et al., 2000; Yang et al., 1999; Mills et al., 1999) and is in contrastto p53-null mice, which are highly tumor prone but lack a developmentalphenotype.
p63
p63 expression is absolutely essential for limb formation and epidermal morphogenesis (integument and tongue) including theformation of adnexa (teeth, hair, mammary and prostate glands,and sweat and lacrimal glands). p63-null animals show severe limb truncations or absence of limbs and absence of skin and craniofacial malformations. They also fail to develop skin and most epithelial tissues (e.g., prostate and mammary glands). The animals do not survive beyond a few days postnatally. Reminiscentof the knockout phenotype in mice, heterozygous germ line pointmutations of p63 in humans cause six rare autosomal dominantdevelopmental disorders
Importantly, basal cells of normal human epithelium including the epidermis strongly express p63 proteins, predominantly theΔNp63 isotype (ratio is 100:1 of ΔNp63 to TAp63; Yang et al., 1998), butlose them as soon as these cells withdraw from the stem cellcompartment (Pellegrini et
al., 2001). Consistent with this notion, keratinocyte differentiation is associated with the disappearance of ΔNp63α(Parsa et al., 1999; Nylander
et al., 2000; Westfall et al., 2003), whereas the expression of p53 target
genes p21 and14-3-3σ, mediating cell cycle arrest, increase. p63 binds p21 and 14-3-3σ promoters and represses them. p63 is also indispensablefor the differentiation of a transitional urothelium and is expressed in normal bladder urothelium. p63 is lost in mostinvasive bladder cancers (Urist et al., 2002).
Together, these data clearly establish a fundamental role of p63 in epithelial stem cell biology and in the apical ectodermalridge of the limb bud, where p63-expressing cells create a signalingcenter (Pellegrini et al., 2001). Whether this role is one in stem cell self-renewal or in stem cell differentiation into stratified epithelium remains a matter of controversy (Yang et al., 1999; Mills et al., 1999). In one model, p63 is requiredfor the ectoderm to commit to epidermal lineages (Yang et al., 1999; Mills et al., 1999), whereas,in the other model, p63 is not required to commit but to
maintainthe stem cell pool and prevent it from differentiation (Brunner et
al., 2002). p73
p73 also has distinct developmental roles. p73 expression is required for neurogenesis of specific neural structures,for pheromonal signaling, and for normal fluid dynamics of cerebrospinal fluid (Yang et al., 2000). The hippocampus is central to learning and memoryand continues to develop throughout adulthood. p73-null animalsexhibit hippocampal dysgenesis due to the selective loss of large bipolar neurons called Cajal-Retzius in the marginal zoneof the cortex and the molecular layers of the hippocampus. These Cajal-Retzius neurons are responsible for cortex organization and coexpress ΔNp73 and the secretory glycoprotein reelin. Inaddition, p73-null mice have severe malformations of the limbic telencephalon. They also suffer from hydrocephalus ( 20%) probably due to hypersecretion of cerebrospinal fluid by the choroid plexus and from a hyperinflammatory response (purulent but sterileexcudates) of the respiratory mucosa likely due to mucus hypersecretion. Moreover, the animals are runted and show abnormal reproductive and social behavior due to defects in pheromone detection. Thelatter abnormality is due to a dysfunction of the vomeronasal organ, which normally expresses high levels of p73.
Role of
Δ
Np73 in Mouse DevelopmentΔNp73 is the predominant form in the developing mouse brain andmight act as a repressor (Yang et al., 1998; Pozniak et al., 2000). In situ hybridization revealsstrong ΔNp73 expression in E12.5 fetal mouse brain in the preplate layer, bed nucleus of stria terminalis, choroid plexus, vomeronasalarea, and preoptic area (Yang et al., 2000). Moreover, ΔNp73 is the only form of p73 found in mouse brain and the sympathetic superior cervical ganglia in P10 neonatal mice (Pozniak et al., 2000). Functional studies and knockout mice showed that ΔNp73 plays an essential antiapoptotic role in vivo. ΔNp73 is required to counteract p53-mediated neuronal death during the normal "sculpting" of the developing mouse neuronal system (Pozniak et al., 2000). Withdrawal of nerve growth factor, an obligate survival factor for mouse sympathetic neurons, leads to p53
neurons are rescued from Adp53-mediated neuronal death by coinfected AdΔNp73. In pull-down assays, mixed protein complexes of p53/ΔNp73 were demonstrated,suggesting one biochemical basis for transdominance in addition to possible promoter competition. Together, these data firmlyput ΔNp73 downstream of nerve growth factor in the nerve growth factor survival pathway. It also explains why p73–/– mice, missing all forms of p73 including protective ΔΝπ73, undergoaccelerated neuronal death in postnatal superior cervical ganglia(Pozniak et al., 2000).
In tissue culture models, p73 also plays a role in differentiationof several cell lineages. p73 expression increases during retinoic acid–induced and spontaneous differentiation of neuroblastomacells (De Laurenzi et al., 2000; Kovalev et al., 1998). In addition, ectopic TAp73β but not p53 induce morphologic and biochemical markers of neuroblastomadifferentiation (De Laurenzi et al., 2000). Moreover, expression of specific COOH-terminal isoforms correlates with normal myeloid differentiation. p73αand p73β are associated with normal myeloid differentiation,whereas p73γ, p73δ, p73ε, and p73θ are associated with leukemicblasts. In fact, p73ε is specific for leukemic blast cells (Tschan et al., 2000).Similarly, TAp73γ and TAp73δ may play a role in the terminal differentiationof human skin keratinocytes (De Laurenzi et al., 2000). This suggests a p73-specificdifferentiation role that is not shared by p53 and, for themost part, not shared by p63 either. p53 has an important developmentalrole in early mouse embryogenesis (E7-8d) as revealed when theautoregulatory feedback loop with MDM2 is removed and p53 levelsremain uncontrolled (Montes et al., 1995; Jones et al., 1995). Nevertheless, in stark contrast to p63- and p73-null mice, p53-null mice make it through development with essentially no problems (with the exceptionof rare exencephaly in females; Donehower et al., 1992; Jacks et
al., 1994).
p63 and p73 expression in normal human tissues
p73 gene expression occurs at very low levels in all normalhuman tissues studied (Kovalev et al., 1998; Ikawa et al., 1999), making detection difficult. p63, mainly its ΔN form, occurs at higher levels and is readilydetectable at the protein level. In embryonic epidermis, p63is the molecular switch for initiation of an epithelial stratification program (Koster et al., 2004). In postnatal epidermis, p63 expression is restrictedto the nuclei of basal cells of normal epithelia (skin, esophagus, tonsil, prostate, urothelium, ectocervix,
and vagina) and tocertain populations of basal cells in glandular structures ofprostate, breast, and bronchi (Yang et al., 1999, Di Como et al., 2002). Specifically, p63 is expressed in myoepithelial cells of the breast and consideredto be the specific marker for those cells in normal breast tissue (Ribeiro-Silva et al., 2003; Reis-Filho et al., 2003). p63 expression in prostate is restricted to basalcells, making it an excellent diagnostic marker in prostate cancer. The vast majority of prostate cancers and preinvasive prostate intraepithelial neoplasia lesions have lost p63 expression.Basal cells play important roles in differentiation and carcinogenesis of the prostate (Davis et al., 2002; Garraway et al., 2003).
Transcriptional and apoptotic activity of p63 and p73
In general, many functional parallels are found among p53, TAp73, and TAp63 on the one hand and among ΔNp73 and ΔNp63 on the otherhand. When ectopically overexpressed in cell culture, p73α and p73β closely mimic the transcriptional activity andbiological function of p53. p73β and, to a lesser extent, p73α bind to canonical p53 DNA binding sites and transactivatemany p53-responsive promoters (Jost et al., 1997; Di Como et
al., 1999; Zhu et al., 1998; Lee et al., 1999), although relative efficiencieson a given p53 target promoter may differ from p53 and also differ among various COOH-terminal isoforms of TAp73 and TAp63(Zhu et al., 1998; Lee et al., 1999). In reporter assays, p73-responsive promoters include well-known p53 target genes involved in antiproliferative and proapoptotic cellular stress responses such as p21WAF1, 14-3-3σ,GADD45, BTG2, PIG3 (Zhu et al., 1998), ribonucleotide reductase p53R2 (Nakano et al., 2000),and IGFBP3 (Steegenga et al., 1999). Bax transactivation is controversial (Zhu et
al., 1998, Steegenga et al., 1999).TAp73α and TAp73β also induce MDM2. Conversely, ectopicp73 overexpression leads to transcriptional repression of vascular endothelial growth factor, analogous to the ability of p53 to transcriptionally suppress vascular endothelial growth factor(Salimath et al., 2000). Although there are probably still dozens of common targetsthat have not yet been described or discovered, it will be important to identify p63/p73-preferred or p63/p73-specific targets. Forexample, Fontemaggi et
BAX p53AIP1 PUMA p21 Mdm2 Cyclin G mp53 ApoptosisImpaired p73 p73 p73 B p73
DNA damage BAX
p53AIP1 PUMA Apoptosis Ac P p73 P p73 CHK1 YAP ? p73 p73 p73 Pin 1 P p73 p300 c-Abl Tyr 99/121 Ac P p73 CHK1 Apoptosis ASPP A p53 DNA damage BAX p53AIP1 PUMA Apoptosis p53 p53 p53 p53 P P Ser 15 Ser 20 ATM ATR CHK1 CHK2 p53 P P P Ser 46 Ac p300 HIPK2 Pin-1 Ser 20 Ser 15 ASPP
Fig.2. p53 family members and DNA damage-induced apoptosis.
(A) In response to DNA damage, p53 is stabilized, phosphorylated at specific serine and threonine residues and acetylated. These post-traslational modifications result in the potentiation of p53-mediated apoptosis.
(B) In response to DNA damage, p73 is stabilized, phosphorylated in specific tyrosine, serine and threonine residues and acetylated. Both c-Abl-mediated tyrosine phosphorylation of p73 and the recruitment of selective coactivators could define the selectivity of p73-mediated apoptosis. Mutant p53 can act as an inhibitor of p73 by the formation of protein complexes comprising mutant p53 and p73.
or p73α promotes the transcriptional activation or repression of common as well as quite distinct patterns of direct target genes (Fontemaggi et al., 2002). Moreover, 14 novel target genes that are differentially regulated by various p53 family members were recently identified (Chen et al., 2003). A surprising "essential cooperativity" among family members for transcriptional function was recently found. In response to DNA damage, induction of p21WAF1 (mediating cell cycle arrest)occurred normally in p63–/– and p73–/– single null mouse embryo fibroblasts (MEFs) and p63/p73–/– double null MEFs. However, in double null MEFs, the induction of Bax, Noxa, and PERP genes (thought to mediate apoptosis) was suppressed. Chromatin immunoprecipitation assays confirmedthat there is no binding of p53 to the Bax, PERP, and NOXA promotersin the absence of p63 or p73, whereas, conversely, p63 still binds to them in p53–/– single null MEFs. Thesedata demonstrate that either p63 or p73 are essential for p53-induced apoptosis (Flores et al., 2002). Ectopic p73 promotes apoptosis in human tumorcell lines independent of their p53 status (Kaghad et al., 1997; Jost et
al., 1997). In fact,in a subset of cancer cell lines, p73β is more efficientin inducing apoptosis than p53 itself (Ishida et al., 2000). Potency differences exist among the COOH-terminal isoforms. Overexpression of p73α,p73β, and p73δ suppresses focus formation of p53-deficientSaos-2 cells, whereas p73γ fails or suppresses only very poorly (Jost et al., 1997; Ishida et al., 2000; De Laurenzi et al., 1998). Similarly, TAp63α lacks significant transcriptionaland apoptotic ability, whereas TAp63γ is very potent in both (Yang et al., 1999).
Regulation of p73 protein stability and transcriptional activity Proteasomes are mediating the turnover of p73 proteins because proteasome inhibitors stabilize p73 isoforms (Balint et al., 1999). In sharp contrast to p53, however, p73 degradation is not mediatedby MDM2. The molecular basis for the MDM2 resistance of p73was found by systematic motif swapping. Region 92-112 of p53, which is absent in p73, was identified to confer MDM2 degradability to p53 (Gu et al., 2000). p73 protein is also resistant to human papillomavirus(HPV) E6, which together
potentialconsequence of the differential MDM2 sensitivity between p53and p73 was seen in tissue culture: ectopic coexpression of p73 leads to a selective decrease of ectopic p53 and endogenousinduced p53 because p53 is susceptible to MDM2, whereas p73 is not (Wang et al., 2001). This suggests a potential down-modulation of p53 by high levels of TAp73 (because MDM2 is also a p73 target),an interesting family twist to keep in mind with respect totumor formation. On a transcriptional level, however, the negativefeedback regulation between the two genes is preserved. MDM2 is transcriptionally activated by p73 and in turn negatively regulates the transcriptional ability of p73, just as it functionstoward p53 (Balint et al., 1999; Dobbelstein et al., 1999; Zeng et al., 1999). However, the mechanism is again distinctfrom p53. The binding to MDM2 causes the disruption of physical and functional interaction with p300/cAMP-responsive element binding protein by competing with p73 for binding to the NH2terminus of p300/cAMP-responsive element binding protein (Zeng et al., 1999). Recently, Rossi et al. found that a HECT-type E3 ubiquitin protein ligase Itch interacts with p73 through the WW protein–protein interaction domains of Itch and the p73 region containing the PY motif, and p53 which does not contain the PY motif fails to interact with Itch (Rossi et al., 2005). According to these results, Itch had an ability to ubiquitinate and degrade p73. Upon DNA damage induced by chemotherapeutic drugs including cisplatin, doxorubicin or etoposide, the endogenous expression levels of Itch were significantly down-regulated through an unknown mechanism, thereby increasing the stability and activity of p73. On the other hand, it has been demonstrated that a novel HECT-type E3 ubiquitin protein ligase NEDL2 directly binds to p73, and this interaction is mediated by the WW domains of NEDL2 and the COOH-terminal region of p73 containing the PY motif (Miyazaki et al., 2003). Unexpectedly, NEDL2 promoted the ubiquitination of p73 in cells, however, NEDL2-mediated ubiquitination increased the stability of p73 and enhanced the p73-dependent transcriptional activation, indicating that there exists a non-proteolytic regulatory role of ubiquitination. Other studies demonstrated that the NH2-terminally truncated form of p73 (ΔNp73) is much more stable than TAp73, suggesting that p73-mediated transcriptional activation is required for the rapid turnover of p73, and that, like p53, one or more transcriptional targets of p73 might promote its proteolytic degradation (Wu et al., 2004). Additionally, Toh et al. reported that c-Jun increases the stability of p73 without direct interaction, and c-Jun-mediated stabilization of p73 is regulated in its transactivation function-dependent manner (Toh et al., 2004). Alternatively, several lines of
evidence suggest that the proteolytic degradation of p73 is regulated in a ubiquitination-independent manner. For example, Ohtsuka et al. found that cyclin G, one of the direct transcriptional targets of p53 and p73, interacts with p73 and induces the latter's rapid degradation (Ohtsuka et al., 2003). According to these results, cyclin G-mediated degradation of p73 was not associated with an increase in its ubiquitination levels. Recently, it has been demonstrated that a U-box-type E3/E4 ubiquitin protein ligase UFD2a interacts with p73 through its COOH-terminal SAM domain, and induces the proteasomal turnover of p73 (Hosoda et al., 2006) Intrinsic E3/E4 ubiquitin protein ligase activity was not necessary for the UFD2a-mediated proteolytic degradation of p73, and UFD2a failed to increase the ubiquitination levels of p73. Similar to Itch, the intracellular expression levels of UFD2a were significantly down-regulated at protein levels in response to cisplatin, thereby leading to a dissociation of free active p73 from the p73/UFD2a complex. Although the precise molecular mechanisms underlying the proteasome-dependent degradation of p73 mediated by UFD2a are not yet known, it is likely that p73 might be recruited to the proteasome through its interaction with UFD2a.
The NAD(P)H:quinone oxidoreductase-1 stabilizes p73α (as wellas p53) but not p73β by binding of its SAM domainto NQO1, which protects p73α from 20S proteasomal degradation that is independent of MDM2. This NQO1-mediated stabilizationof p73α and p53 provides one explanation why NQO1 knockout micehave a cancer phenotype and humans with inactive NQO1 polymorphismsare susceptible to cancer (Asher et al., 2002).
In addition to post-translational modifications including phosphorylation and acetylation, the activity of p73 is regulated by physical interaction with several viral and cellular proteins. Like p53, p73 was associated with the adenovirus E1A and the T-cell lymphotropic virus I-derived Tax, and these interactions inhibited the activity of p73 (Irwin et al., 2001). On the other hand, the viral proteins which can bind to and inactivate p53, including the adenovirus E1B, papilomavirus E6 and simian virus 40 T antigen, failed to interact with p73 (Marin et al., 1998; Roth et al., 1998; Steegenga et al., 1999). For cellular proteins, MDM2 interacted with both p53 and p73, and inactivated their activities (Zeng et al., 1999; Dobbelstein et al., 1999).
p73 but extended its half-life, thereby enhancing its transcriptional activation. Itch promoted the ubiquitination-mediated proteasomal turnover of p73. YAP interacted with the PY motif of p73 through its WW domain, and stimulated p73-mediated transcriptional activation. Nakagawara’ lab performed a conventional yeast-based two-hybrid screening using the extreme COOH-terminal tail of p73 not found in p53. Finally, they identified the c-Myc-binding protein (MM1), RACK1 and RanBPM as p73-binding proteins (Kramer et al., 2005; Watanabe et al., 2002; Ozaki et al., 2003). Based on these results, MM1 attenuated the c-Myc-mediated inhibition of transcriptional activity of p73, whereas RACK1 significantly inhibited the function of p73 and its inhibitory effect was counteracted by pRB. RanBPM increased the stability of p73 by reducing its ubiquitination levels. The proteins identified had no detectable effects on p53. By using a new CytoTrap yeast two-hybrid screening, they identified the protein kinase A catalytic subunit β (PKA-Cβ) as a novel binding partner of p73 (Hanamoto
et al., 2005). PKA-Cβ bound to both the NH2- and COOH-terminal regions of p73, and inhibited its transcriptional activity. PKA-Cβ efficiently phosphorylated p73, and PKA-Cβ-mediated inhibition of p73 was dependent on the kinase activity of PKA-Cβ. These observations strongly suggest that the regulatory mechanisms of p73 are distinct from those of p53.
The ankyrin-rich, Src holomogy 3 domain, proline-rich proteinsASPP1 and ASPP2 stimulate the apoptotic function of p53, p63, and p73 (Bergamaschi et al., 2004; Samuels-Lev et al., 2001). By binding to the DBD of p53, p63, and p73,ASPP1 and ASPP2 stimulate the transactivation function of allthree proteins on the promoters of Bax, PIG3, and PUMA but not MDM2 or p21WAF-1/CIP1. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members.
Post-translational modifications during activation
p53 stabilization and activation by genotoxic stress is associated with multiple post-translational modifications at the NH2 andCOOH termini of p53 in vivo. In close temporal relationship to stress, the NH2 terminus undergoes heavy phosphorylation(Ser15, Ser20, Ser33, Ser37, Ser46, Thr18, and Thr81), whichis thought to stabilize the protein by interfering with MDM2 binding, thereby disrupting the constitutively targeted degradation. The COOH terminus also undergoes site-specific phosphorylation (Ser315 and Ser392), acetylation (Lys320, Lys373, and Lys382), and sumoylation (Lys386).
The COOH-terminal modifications arethought to activate the transcriptional activity of p53 (Appella et al., 2001).So-called stress kinases (e.g., ATM, ATR, and Chk2), which detect genotoxic stress and initiate signal transduction, are in vivokinases for specific p53 serine residues, whereas the histone acetyltransferases p300/cAMP-responsive element binding protein and PCAF (which at the same time are transcriptional coactivators)acetylate p53.
Recent studies revealed that p73 is induced to be accumulated in response to a subset of DNA-damaging agents, including cisplatin, adriamycin, camptothecin and etoposide (Irwin et al., 2003). p73 is predominantly regulated at the post-translational level. Accumulating evidence strongly suggests that chemical modifications of p73, such as phosphorylation and acetylation, prolong its half-life, which, in turn, enhance its transcriptional and pro-apoptotic activity. During the cisplatin-mediated apoptotic process, p73 is phosphorylated at Tyr-99 and stabilized in a pathway dependent on nuclear non-receptor tyrosine kinase c-Abl (Gong et al., 1999; Agami et al., 1999; Yuan et al., 1999). In addition to c-Abl, exposure to cisplatin promoted a complex formation between p73 and a protein kinase Cδ catalytic fragment, which phosphorylated p73 at Ser-289 and increased its stability and transcriptional activity (Ren et al., 2002). Recently, it has been shown that cisplatin-induced apoptosis is associated with p73 phosphorylation at Ser-47 mediated by Chk1 (Gonzalez et al., 2003). Chk1-dependent phosphorylation resulted in an increase in the transcriptional activity of p73. In contrast, CDK-mediated phosphorylation of p73 led to significant inhibition of its transcriptional activity (Gaiddon et al., 2003) indicating that the phosphorylation of p73 might not always convert a latent form of p73 to an active one. Alternatively, p73 is regulated by acetylation. p73 was previously found to be associated with p300 histone acetyltransferase through its NH2-terminal transactivation domain, and this interaction resulted in a significant enhancement of p73-mediated transcriptional activation as well as apoptosis (Zeng et al., 2000). Costanzo
et al. reported that p300 acetylates p73 at Lys-321, Lys-327 and Lys-331 in
response to doxorubicin in a c-Abl-dependent manner, and the acetylated forms of p73 have pro-apoptotic activity (Costanzo et al., 2002). Intriguingly, the p300-mediated acetylation of p73 was stimulated by prolyl
Sumoylation of COOH-terminal Lys627 occurs specificallyin p73 but not in p73ß in vitro. However, in contrast to sumoylation of p53, which activates its transcriptional activity, sumoylation of p73 promotes its degradation (Minty et al., 2000).
Alteration of p73 expression in human cancer
p73 maps to chromosome 1p36.33, which frequently undergoes lossof heterozygosity in breast and colon cancer, neuroblastoma, oligodendroglioma, and melanoma. This fact, in conjunction with the functional similarity to p53, originally led to the proposalthat p73 is a tumor suppressor gene (Kaghad et al., 1997). Genetic data on mostcancer types (with the notable exception of leukemias and lymphomas),however, exclude p73 as a classic Knudson-type tumor suppressor, which by definition is targeted to undergo loss of expressionor function during tumorigenesis. To date, in a total of >1,100primary tumors, loss of function mutations in p73 are vanishingly rare (0.6%). Surprisingly, the most common identifiable cancer-specific alteration is overexpressionof various isoforms of the wild-type p73 rather than a lossof expression (Kaghad et al., 1997). This suggests that p73 plays an oncogenicrole in tumorigenesis. The single exceptions to this picturemight be lymphoid malignancies and, possibly, bladder cancer. Although overexpression of p73 gene was found in B-cell chronic lymphocytic leukemia (Novak et al., 2001) and during differentiation of myeloid leukemic cells (Tschan et al., 2000), p73 has been found to be transcriptionallysilenced in some lymphoblastic leukemias and lymphomas due tohypermethylation (Corn et al., 1999; Kawano et al., 1999). Likewise, based on one immunocytochemicalstudy with prognostic analysis, invasive high-grade bladder cancers, which had lost p73 (and p63) staining, had a poorerclinical outcome (Puig et al., 2003).
To date, significant prevalence of p73 overexpression has beenfound in 12 different tumor types including tumors of breast (Zaika et al., 1999), neuroblastoma (Kovalev et al., 1998), lung (Mai et al., 1998; Tokuchi et al., 1999), esophagus (Cai et al., 2000), stomach (Kang et al., 2000), colon (Sunahara et al., 1998), bladder (Chi et al., 1999; Yokomizo et al., 1999), ovarian cancer (70%of cases in one cohort; Ng et al., 2000; Chen et al., 2000; Zwahlen et al., 2000), ependymoma (Zwahlen et al., 2000), liver cancer (Tannapfel et al., 1999a), cholangiocellular carcinoma (Tannapfel et
myelogenous leukemia (Tschan et al., 2000; Peters et al., 1999), colon carcinoma (Guan et al., 2003; Sun et al., 2002), and head and neck squamous carcinoma(associated with distant metastasis; Choi et al., 2002; Weber et al., 2002a; Weber et al., 2002b). Most studies measure overexpression of full-length p73 mRNA (TAp73) by reverse transcription-PCR, but a few studies also measure overexpressionof TAp73 protein(s) by either immunoblot or immunocytochemistry. For example, there is overexpression of TAp73 transcripts (5-to 25-fold) in 38% of 77 invasive breast cancers relative to normal breast tissue and in five of seven breast cancer cell lines (13- to 73-fold; Zaika et al., 1999). Likewise, there is overexpressionof TAp73 transcripts in a subset of neuroblastoma (8- to 80-fold)and in 12 of 14 neuroblastoma cell lines (8- to 90-fold; Kovalev et al., 1998). A close correlation between p73 mRNA levels and proteinlevels was shown in ovarian carcinoma cell lines (Ng et al., 2000). In aseries of 193 patients with hepatocellular carcinoma, 32% of tumors showed detectable (high) p73 by immunocytochemistry andin situ hybridization, whereas all normal tissue had undetectablelevels (low; Tannapfel et al., 1999a). Of note, primary tumors and tumor cell lines with p73 overexpression tend to simultaneously overexpress a complex profile of shorter COOH-terminal splice variants (p73γ,p73δ, p73ε, and p73φ), whereas the normal tissue of origin is limited to the expression of p73α and p73β (Zaika et al., 1999). Importantly,patients with high global p73 protein expression had a worse survival than patients with undetectable levels (Tannapfel et al., 1999a; Sun
et al., 2002).
There is an emerging sense that the dominant-negative ΔTAp73isoforms rather than TAp73 might be the physiologically relevant components of tumor-associated p73 overexpression, functionally overriding an often concomitant increase in TAp73 expression.This might have escaped notice because many of the early p73 overexpression studies in human cancers determined total p73 levels (all isoforms). Therefore, up-regulation of ΔTAp73 forms likely contributed to the elevated total p73 levels found previouslyin human cancers. Although, to date, only a few limited studiesof tumors (breast cancer, gynecologic cancers, hepatocellular carcinoma, and neuroblastoma) focused on ΔTAp73, highly prevalent, tumor-specific up-regulation of ΔNp73 or ΔN'p73 (producing thesame protein) has already
ΔNp73 overexpression appears to have a clinical impact at least in some cancer types. ΔNp73 was foundto be an independent prognostic marker for reduced progression-free and overall survival in neuroblastoma patients (Casciano et al., 2002).
Alteration of p63 expression in human cancer
p63 is not a tumor suppressor. The analysis of p63 in cancersof patients with germ line mutations or somatic mutations indicates similar lack of mutations but up-regulation of dominant-negative forms. For example, no p63 mutations were found in 47 bladdercancers (Park et al., 2000) or 68 squamous cell carcinoma of the head andneck (Weber et al., 2002). Only 1 missense mutation (Ala148Pro) of 66 varioushuman tumors and 2 missense mutations in 35 tumor cell lineswere found.
The human p63 gene is on chromosome 3q27-28 within a regionthat is frequently amplified in squamous cell, cervical, and prostate carcinomas. Some lung cancers and squamous cell carcinomasof the head and neck show p63 overexpression associated witha modest increase in p63 copy numbers (Hibi et al., 2000). In 25 primary nasopharyngeal carcinomas, alltumor cells overexpressed predominantly ΔNp63, which in normal nasopharyngeal epithelium is limited to proliferating basaland suprabasal cells (Crook et al., 2000). In esophageal squamous cell carcinoma,ΔNp63 is the major isotype expressed throughout. In contrast, in normal esophagus, p63 staining is restricted to the basaland suprabasal cell layers (Choi et al., 2002, Hu et al., 2002). Thus, the maintenanceof the ΔNp63 isoforms in squamous cancers may contribute to keepingthe cells in a stem cell–like phenotype, thereby promotingtumor growth. Up-regulation of ΔNp63 was also found in 30 of 47 bladder cancers (Park et al., 2000). Interestingly, TAp63 was concomitantly down-regulatedin 25 of those 47 tumors.
Transcriptional regulation of the main promoter of p73
It has been recently established that the cellular and viral oncogenes E2F1, c-Myc, and E1A can induce and activate the endogenousTAp73α and TAp73β proteins for target gene transactivation, apoptosis, and growth suppression in p53-deficient human tumorcells (Zaika et al., 2001; Stiewe et
E2F1 transcription factor plays an important role in the regulation of cell cycle progression by inducing the transcription of genes whose products are directly or indirectly required for entry into the S phase (Johnson et al., 1993). In addition to the proliferative effect of deregulated E2F1 activity, unscheduled E2F1 activation leads to apoptosis to protect cells from cellular transformation (Shan et al., 1994). Consistent with this notion, E2F1-deficient mice exhibited a high incidence of unusual tumors (Yamasaki et
al., 1996; Field et al., 1996). E2F1-induced apoptosis is regulated in a
p53-dependent or p53-inp53-dependent manner. It is interesting that the p73 promoter region contains a TATA-like box and at least three E2F1-binding sites, and indeed the enforced expression of E2F1 strongly stimulates the transcription of p73 through the direct binding to the E2F1-responsive elements in the p73 promoter (Irwin et al., 2000; Stiewe et al., 2000). The E2F1-mediated up-regulation of p73 results in a significant induction of apoptosis. Alternatively, E2F1 might also contribute to the up-regulation of p73 mRNA levels during muscle and neuronal differentiation of murine C2C12 myoblasts and P19 cells, respectively (Fontemaggi et al., 2001). In addition to E2F1, cellular and viral oncogene products such as c-Myc and E1A indirectly activated the transcription of p73 (Zaika et al., 2001).
Becauseoncogene deregulation of E2F1 and c-Myc are one of the most common genetic alterations in human tumors, these findings mightprovide a physiologic mechanism for TAp73 overexpression in tumors. Taken together, these data establish another importantlink between p73 and human cancer.
p73 is required for antigen-induced death of circulating peripheralT cells after T-cell receptor activation and for tumor necrosisfactor-α-induced death of thymocytes (immature T cells). Thisdeath pathway is mediated via the E2F1-p73 (Lissy et al., 2000; Wan et al., 2003). Conversely,the survival of antigen-stimulated T cells requires nuclearfactor kB–mediated inhibition of p73 expression (Wan et al., 2003).Consistent with this notion, E2F1-null mice exhibit a markeddisruption of lymphatic homeostasis with increased numbers of T cells and splenomegaly, suggesting that p73 plays a role in tumor surveillance pathways of lymphoid cells (Yamasaki et al., 1996; Field
et al., 1996). Moreover,the p73 gene is transcriptionally silenced in acute lymphoblastic leukemia and Burkitt's lymphoma due to hypermethylation
lymphomas of the mouse, the p73 locus undergoes lossof heterozygosity in 33% of the cases (Herranz et al., 1999). Thus, in lymphoid tumors, p73 shows some genetic features of a classic tumor suppressor gene. Early growth response factor-1, an immediate early gene that is activated by mitogens in quiescent postmitotic neurons, induces apoptosis in neuroblastoma cells. This apoptosis seemsto be mediated by p73, which is elevated in cells overexpressingearly growth response factor-1 (Pignatelli et
al., 2003).
Recently, Fontemaggi et al. identified a 1 kb negative regulatory fragment within the first intron of p73 gene (Fontemaggi et al., 2001). This intronic fragment significantly reduced the activity of the p73 promoter upon E2F1 overexpression. Of note, the p73 intronic fragment contained six consensus binding sites for transcriptional repressor ZEB. Ectopic expression of ZEB in C2C12 myoblasts attenuated myotube formation, and repressed the transcription of p73. In accordance with these results, the dominant negative form of ZEB had an ability to restore the expression levels of p73 in proliferating cells.
Because DNA hypermethylation contributes to the alteration of the entry of transcription factors into the regulatory region, the epigenetic modification of the p73 promoter region through aberrant hypermethylation could be an alternative molecular mechanism for silencing the p73 gene. Corn et al. described the aberrant promoter methylation of p73 as occurring frequently in primary acute lymphoblastic leukemias and Burkitt's lymphomas, whereas the p73 promoter methylation was not detected in normal lymphocytes or bone marrow (Corn et al., 1999). Similar results were also reported by Kawano et al (Kawano et al., 1999). In contrast, hypermethylation of the p73 promoter region was not observed in solid tumors including breast, renal, colon cancers or neuroblastomas (Banelli et
al., 2000), suggesting that the methylation-dependent silencing of p73
transcription might be specific to hematological malignancies. Mutual regulation between p73 and p53
Previously, it has been shown that tumor-derived p53 mutants but not wild-type p53 interact with p73, and abrogate its function (Di Como et al., 1999). Subsequent studies demonstrated that the ability of p53 mutants to interact with p73 depends on the nature of the p53 mutations as well as the polymorphism at codon 72 (Pro-72 or Arg-72) of p53 mutants (Marin et al.,
2000), in particular, p53 mutants carrying Arg-72 bound to p73 better than p53 mutants with Pro-72. Consistent with this notion, p53 mutants carrying Arg-72 act as more potent inhibitors of chemotherapy-induced apoptosis than the p53 mutants with Pro-72 (Bergamaschi et al., 2003). Functionally, formation of such stable complexesleads to a loss of p73- and p63-mediated transactivation and proapoptotic abilities. Moreover, E2F1-induced p73 transactivation, apoptosis, and colony suppression was inhibited by coexpressed p53His175 (Stiewe et al., 2000). It suggests thatin tumors that express both TAp73 and mutant p53 (typicallyat very high levels due to deficient MDM2-mediated degradation),the function of TAp73 and TAp63 might be inactivated. This gain-of-function results in increased tumorigenicitycompared with p53-null parental cells, increased resistanceto cancer agents, and increased genomic instability due to abrogation of the mitotic spindle checkpoint (Dittmer et al., 1993; Shaulsky et al., 1991; Halevy et al., 1990). Other studies focused on the functional interaction between wild-type p53 and p73. Miro-Mur et al. reported that p73 induces both accumulation and activation of wild-type p53 by preventing MDM2-mediated degradation through MDM2 titration (Miro-Mur et al., 2003). In addition, Goldschneider et al. found that p73 promotes the nuclear localization of wild-type p53 in neuroblastoma cells in which p53 is predominantly expressed in cytoplasm (Goldschneider et al., 2003). These results suggest that p73 has an ability to enhance the activity of wild-type p53. In contrast, Vikhanskaya et al. described that p73 reduces the p53-mediated transcriptional activation through the competition of the same DNA-binding site (Vikhanskaya et al., 2000). These controversial results regarding the effects of p73 on wild-type p53 might be at least in part due to the different cell systems used in those studies.
Recently, it has been shown that p53-dependent apoptosis requires the indirect contribution of at least one other p53 family member, p73 or p63 (Flores et al., 2002). Thus, it is likely that p73 cooperates with p53 to promote apoptotic cell death. These findings emphasize the functional importance of p73 in the regulation of the DNA damage-induced apoptotic response.
An autoregulatory feedback loop exists among p53, Tap73 and ΔNp63
p53 and TAp73 regulate ΔNp73 but not ΔNp63 levels by binding tothe p73 P2 promoter and inducing its transcription. A p73-specificresponsive element was mapped within the P2 region (Nakagawa et al., 2002). This generates a negative feedback loop analogous to the p53-MDM2loop that in turn negatively regulates the activity of p53 andp73 (Nakagawa et al., 2002; Kartasheva et al., 2002; Vossio et al., 2002; Grob et al., 2001). ΔNp73 blocks p53 and TAp73 activitythrough heterocomplex formation (Stiewe et
al., 2002; Zaika et al., 2002; Nakagawa et al., 2002) or through promoter
competition (Stiewe et al., 2002; Kartasheva et al., 2002;) and thus contributes to the terminationof the p53/p73 response in cells that do not undergo apoptosis. In contrast to ΔNp73, ΔNp63 expression is transcriptionally repressedby p53 (Waltermann et al., 2003).
p73 and chemosensitivity
Endogenous p73 protein levels increase in response to cisplatin and Adriamycin (Agami et al., 1999; Costanzo et al., 2002; Gong et al., 1999). Although originally thought torespond only to a limited spectrum, it is now clear that TAp73 (α more than β) is induced by a wider variety of chemotherapeutic agents (Adriamycin, cisplatin, taxol, and etoposide) in differenttumor cell lines (Irwin et al., 2003; Bergamaschi et al., 2003). p73 accumulation is due to increased transcription and increased protein stabilization and leads to induction of apoptotic target genes such as apoptosis-induced protein-1. Conversely, blocking TAp73 function (either by theinhibitory p73DD fragment or by p73 small interfering RNA) leadsto enhanced chemoresistance, which is independent of the p53gene status. Of note, whereas the presence of p73 is essentialfor p53 to induce apoptosis in fibroblasts (Flores et al., 2002), p73 on theother hand can induce apoptosis in cells that lack functional p53 (Irwin et al., 2003). This confirms the importance of p73 in the responseto chemotherapeutic agents (Bergamaschi
et al., 2003).
In cell culture, overexpression of antiapoptotic p73 isoforms can also block chemotherapy-induced apoptosis in wild-type p53tumor cells (Zaika
et al., 2002; Vossio et al., 2002). Moreover, overproduction of certainp53 mutants can block p73 function and chemotherapy-induced apoptosis (Di
Como et al., 1999; Gaiddon et al., 2001; Blandino et al., 1999). This effect is most strongly linkedto the Arg72 polymorphism of the p53 gene (Irwin et al., 2003; Marin et al., 2000; Bergamaschi et al., 2003) andis mediated by stable hetero-oligomers involving the DBDs. Bergamaschi et al. have used
different cell lines forced to express a seriesof p53 mutants as either Arg (72R) or Pro (72P) versions atcodon 72. Only Arg mutants correlated with chemoresistance.These data were mirrored in a series of polymorphic head andneck cancer patients with the same p53 mutants: 72R patientsshowed poor response to chemotherapy and shorter survival (Bergamaschi et al., 2003).Conversely, down-modulation of endogenous p53 mutants enhances chemosensitivity in p53-defective mutant cells (Irwin et al., 2003). Consequently, a promising therapeutic approach includes the use of small interferingRNA specifically directed against particular p53 mutants, which might restore chemosensitivity of tumor.
YAP
Large protein complexes that include transcription factors and specific coactivators frequently govern activation of specific sets of genes (Naar et
al., 2001). As in the case of p53, p73 is controlled by interaction partners
and these interactions might determine the extent to that p73 contributes to apoptosis. The existence of the protein complex YAP/p73 was the first evidence linking WW domain containing proteins to the p53 family members (Strano et al., 2001). YAP, the first protein in which a WW domain was identified, is a phosphoprotein of 65 kDa that interacts with the SH3 domain of the c-yes protooncogene product, a nonreceptor tyrosine kinase of the Src family (Sudol, 1994). WW domains are protein-protein interaction modules that recognize short proline-rich motifs of diverse proteins involved in various signaling pathways (Sudol and Hunter, 2000). The name refers to two signature tryptophan (WW) residues that are spaced 20–22 amino acids apart and play an important role in the domain structure and function (Sudol and Hunter, 2000). In addition to a type I WW domain, YAP also contains a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain. A recent work has reported the identification of a 14-3-3 binding molecule, named TAZ (transcriptional coactivator with PDZ binding motif), that shares a remarkable homology with YAP (Kanai et al., 2000). It has recently been shown that YAP is a potent transcriptional coactivator. YAP binds to and coactivates the Runx and the four TEAD/TEF transcription factors (Yagi et
al., 1999 and Vassilev et al., 2001).
A close link between YAP and the transcription factors of the p53 family has recently emerged (Strano et al., 2001 and Basu et al., 2003). We had originally reported that YAP engages in a physical association with p73 and p63 (Strano et al., 2001). In terms of binding to p53 family members, YAP possesses two levels of specificity, binding to long, but not to short, forms of p73 and p63, and it does not bind to p53 at all (Strano et al., 2001). The binding of YAP to p73 or p63 results in a strong transcriptional coactivation (Strano et al., 2001 and Basu et al., 2003; S.S. and G.B., unpublished data). It has been reported that YAP is phosphorylated by AKT, and such modification impairs YAP-nuclear translocation and attenuates p73-mediated apoptosis (Basu et al., 2003).
In a recent work we demonstrated that p73 is required for the nuclear translocation of endogenous YAP in cells exposed to cisplatin and that YAP is recruited by PML into the nuclear bodies (NBs) to promote p73
YAP 1
WW1 ACTIVATIONDOMAIN
SH3 BINDING DOMAIN PDZ BINDING DOMAIN
YAP 2
WW1 WW2 ACTIVATIONDOMAIN
Fig.3. Structure of YAP proteins.
Two YAP isoforms have been identified, characterized by the presence of one (YAP1) or two (YAP2) WW domains. WW domains are protein-protein interaction modules that recognize short proline-rich motifs of diverse proteins involved in various signaling pathways. The name refers to two signature tryptophan (WW) residues that are spaced 20–22 amino acids apart and play an important role in the domain structure and function. In addition to a type I WW domain, YAP also contains a PDZ interaction motif, an SH3 binding motif, and a coiled-coil domain.
transcriptional activity (Strano et al., 2005). We found that YAP contributes to p73 stabilization in response to DNA damage and promotes p73-dependent apoptosis through the specific and selective coactivation of apoptotic p73 target genes and potentiation of p300-mediated acetylation of p73. Indeed, endogenous p73, YAP, and p300 proteins are concomitantly recruited to the regulatory regions of the apoptotic target gene p53AIP1 only when cells are exposed to apoptotic conditions. Silencing of YAP by specific siRNAs impairs p300 recruitment onto the p73 apoptotic target gene
p53AIP1 upon DNA damage, and this correlates with the reduction of
histone acetylation at the same promoter site and delayed or reduced apoptosis (Strano et al., 2005). Altogether, these results identify YAP as an important determinant for p73 target gene specificity through p300 recruitment and p73 acetylation.
Recently, Rossi et al. have shown that Itch, a human ubiquitin-protein ligase, which belongs to the Nedd4-like E3 family containing a WW domain (Perry et al., 1998), binds and ubiquitinates p73 via its PPPY motif and determines its rapid proteosome-dependent degradation in a ubiquitin-dependent manner (Rossi et al., 2005). More recently Levy et al. have shown that YAP competes with Itch for binding to p73 at the PPPY motif and this prevents Itch ubiquitination, and subsequent degradation, of p73 (Levy et al., 2006). Treatment of cells with cisplatin leads to an increase in p73 accumulation and induction of apoptosis, but both were dramatically reduced in the presence of YAP siRNA.
P 14-3-3 YAP Akt p53AIP1 Bax Killer/DR5 p21 Pig3 Apaf-1 p73 X p73 X DNA damage YAP Akt 14-3-3 Apoptosis p73 p73 p73 p73 p73 p21 Pig3 Apaf-1 X p73 Ac PML Killer/DR5 p53AIP1 Bax Ac YAP p300 Ac p73 YAP p73 PML PML NBs P 14-3-3 YAP Akt p53AIP1 Bax Killer/DR5 p21 Pig3 Apaf-1 p73 X p73 X DNA damage YAP Akt 14-3-3 Apoptosis p73 p73 p73 p73 p73 p21 Pig3 Apaf-1 X p73 Ac PML Killer/DR5 p53AIP1 Bax Ac YAP p300 Ac p73 p73 YAP p73 PML PML PML NBs
Fig.4. Model for p73 gene target specificity modulation by YAP in response to DNA damage.
DNA damage causes p73 accumulation, release of YAP from cytoplasmic multiprotein complexes containing 14-3-3 and AKT, and YAP relocalization into the nucleus. PML is required to localize YAP into the NBs to coactivate p73. The interaction with YAP promotes p73 stabilization, binding to p300 and its acetylation. Under apoptotic conditions, the transcriptionally active complex that contains acetylated p73, YAP and p300 assembles onto the regulatory regions of the p53-p73 complex proapoptotic target genes p53AIP1 and BAX.
S100 proteins
The S100 proteins are small acidic proteins (10-12 kDa) that are found exclusively in vertebrates (Schafer et al., 1996). With at least 25 members found to date in humans, the S100 proteins constitute the largest subfamily of the EF-hand proteins. First identified by Moore in 1965 (Moore et al., 1965), the S100 proteins have 25-65% identity at the amino acid level characterized by the presence of two Ca2+ binding sites of the EF-hand type (i.e., helixloop-helix), one of which, located in the S100 N-terminal half, is unconventional, while the other one, located in the S100 C-terminal half, is canonical. As a consequence, Ca2+ binding to individual EF hands occurs with different affinities, a lower affinity in the case of the N-terminal site and a ~ 100-times higher affinity in the case of the C-terminal site. The two EF hands are interconnected by an intermediate region, referred to as the hinge region, and the C-terminal EF hand is followed by a C-terminal extension. S100 members differ from one another mostly for the length and sequence of the hinge region and the C-terminal extension, which are thus suggested to specify the biological activity of individual proteins. Three members of the family, i.e., profilaggrin, trychohyalin, and repetin, are large proteins that exhibit an S100 motif along their primary sequence. With the exception of calbindin D9k, which is monomeric, all other small S100 proteins exist within cells as homodimers in which monomers are related by a twofold axis of rotation and are held together by noncovalent bonds. Upon Ca2+-binding, helix III becomes perpendicular to helix IV, the hinge region swings out, and a cleft forms in each monomer, which is defined by residues in the hinge region, helices III and IV and the C-terminal extension, and is buried in apo S100 monomer. Residues defining this cleft are believed to be important for the Ca2+-dependent recognition of S100 target proteins. The hinge region and the C-terminal extension play a critical role in the interaction of S100A1, S100B, S100A10, and S100A11 with several target proteins (Donato, 2001; McClintock et al., 2002). Thus, upon Ca2+ binding, each S100 monomer opens up to accommodate a target protein (with the exception of S100A10 that is normally in an open-up state), and the S100 dimer can bind target proteins on opposite sides. By this mechanism, an S100 dimer functionally crosslinks two homologous or heterologous target proteins. Given the positions of the helices of one monomer relative to those of the other monomer (particularly helices I and IV and I’ and IV’), helix I’ might participate in the formation of each of the two binding surfaces on a
Fig.5. S100 protein structure and model of S100 protein/target protein interaction.
The overall structure of each S100 protein family member includes four alpha-helical segments, two calcium-binding EF-hands (one non-canonical site binds calcium with low affinity, and one canonical), a central hinge region of variable length, and the C- and N-terminal variable domains.
S100 proteins exist as anti-parallel dimers. An increase in calcium concentration results in a conformation change in the dimer that results in exposure of a cleft, which forms the target protein binding site. Once in the calcium-loaded state, each S100 protein dimer can interact with a target protein via its C-terminal domain. Thus, a single S100 protein dimer can ligate two target proteins.
given S100 dimer (Donato, 2001). This would explain why most S100 members form dimers; one S100 monomer is not enough for binding a target protein or, alternatively, target protein binding to S100 occurs with reduced strength. Probably, dimeric S100 proteins use different mechanisms for interacting with their target proteins, as in some cases Ca2+ is not required for S100 to bind to an effector protein, indicating that residues other than those that become exposed to the solvent upon Ca2+ binding might recognize definite target proteins.
Intracellular roles of S100 proteins
S100 proteins have been implicated in the regulation of protein phosphorylation, the dynamics of cytoskeleton constituents, Ca2+ homeostasis, enzyme activities, transcription factors, cell growth and differentiation, and the inflammatory response (Donato, 1999, 2001; Scha¨ fer and Heizmann, 1996; Zimmer et al., 1995).
Inhibition of protein phosphorylation
Inhibition of protein phosphorylation by S100 proteins depends on blockade of access of kinases to the pertinent protein substrate. This might represent a means to finely tune the activity of a given effector protein since in most cases the inhibitory effect of S100 protein on protein phosphorylation is Ca2+-dependent. Also, this represents an example of cross-talk between (cytosolic) Ca2+-based activities and protein phosphorylation. There are a few examples of S100-dependent inhibition of protein phosphorylation that are potentially relevant: (1) inhibition of caldesmon phosphorylation by S100B results in the reversal of caldesmon-dependent inhibition of actomyosin ATPase activity (Fujii et al., 1990; Pritchard and Martson, 1991; Skripnikowa and Gusev, 1989); (2) inhibition of microtubule (MT)-associated τ protein phosphorylation by S100B has been suggested to be an important mechanism of neuronal protection from τ hyperphosphorylation in Alzheimer’s disease (Yu and Fraser, 2001), in neurons in which S100B is expressed; (3) inhibition of p53 phosphorylation by S100B might result in inhibition of p53-dependent transcription activation via disruption of the p53 tetramer and, hence, of tumor suppressor
activity of p53 (Rustandi et al., 2000; Lin et al., 2001); (4) inhibition of an unknown substrate of protein kinase Cβ might result in inhibition of the hypertrophic response following myocardial infarction (Tsoporis et al., 1997, 1998); (5) inhibition of myosin heavy chain phosphorylation by S100A4 might be linked to modulation of the cytoskeleton dynamics in metastatic cells (Davies et al., 1996; Kriajevska et al., 1998); (6) inhibition of p53 phosphorylation by S100A4 might result in inhibition of p53-dependent transcription activation and, hence, of tumor suppressor activity of p53 (Grigorian et al., 2001); and (7) inhibition of ANXA2 phosphorylation by S100A10 might result the sequestration of ANXA2 in the cytoplasm (Eberhard et al., 2001) and consequent modulation of the activities of ANXA2, a Ca2+-dependent phospholipid-, membrane-, and cytoskeleton-binding protein (Gerke and Moss, 2002).
Regulation of enzyme activity
S100A1 stimulates the sarcomeric, myosin-associated giant kinase twitchin in a Ca2+- and Zn2+-dependent manner in vitro (Heierhorst et al., 1996). Twitchin is a member of a family of giant protein kinases involved in the regulation of muscle contraction and the mechanoelastic properties of the sarcomere in invertebrates. The corresponding vertebrate protein is titin, which was recently shown to interact with S100A1 (Yamasaki et al., 2001). S100B and, to a smaller extent, S100A1 stimulate Ndr, a nuclear serine/threonine protein kinase important in the regulation of cell division and cell morphology, in a Ca2+-dependent manner (Millward et al., 1998). S100B and S100A1 stimulate a membrane-bound guanylate cyclase (GC) activity in photoreceptor outer segments in vitro (Duda et al., 1996; Pozdnyakoz et al., 1997). The S100A8/S100A9 heterodimer modulates the activity of casein kinase I and II, two enzymes that phosphorylate topoisomerase I and RNA polymerases I and II, pointing to a potential role of S100A8 and S100A9 and/or the S100A8/S100A9 heterodimer in the regulation of myeloid cell maturation and function (Lagasse et al., 1988; Murao et al., 1989; Zwadlo et al., 1988). S100A10 inhibits the activity of cytosolic (85-kDa) phospholipase (PL) A2 (Wu et al., 1997).
